The purpose of the proposed research is a detailed investigation into the mechanism of the Alpha-chymotrypsinyl 2-(5-n-alkyl) furoate reactions with water and with aliphatic alcohols. On the basis of previous work we have concluded that the acylated Alpha-chymotrypsin exists in two forms which are rapidly interconvertible, and which both react, but at different rates. We have also proposed the existence of an obligatory reaction intermediate in the transesterification reaction of the acylated enzyme, a proposal based on a time-dependent [16O/18O]KIE. This work is to be continued in order to i) verify the rapid equilibrium model; ii) obtain the rate constants associated with the formation and breakdown of the obligatory intermediate; and iii) extend the KIE measurements to other isotopically substituted atoms. Specifically, I propose the following experiments: measure the temperature dependence of acylated enzyme hydrolysis using chymotrypsinogen, Delta-chymotrypsin and Met-192-sulfoxide chymotrypsin; obtain the CD spectrum of Alpha-chymotrypsinyl 2-(5-nitro) furoate; obtain improved measurements on the temperature dependence of Alpha-chymotrypsin-catalyzed transesterifications, measure the KIE's associated with the substitution of 13C into the carbonyl cargon of the acyl moiety, with the substitution of 18O into the secondary position of the carbonyl oxygen, and to measure the effect of D2O on the primary [16O/18O]KIE in the transesterification reaction. The KIE measurements are to be performed using the method of permselective membrane/mass spectroscopy, a method we are currently developing. We plan to utilize the results obtained in these experiments to determine the activation entropies and enthalpies associated with the rate limiting steps of Alpha-chymotrypsin deacylations, to gain information on the sensitivity of this reaction to proteins conformational changes, and to model the geometries of the transition states involved in the deacylation reactions.